To begin, seed HEK293T cells into a 10-layer Cell Factory containing DMEM high-glucose media with 10%FBS. Place the cells in an incubator at 37 degrees Celsius under 5%carbon dioxide supplementation overnight. Next, aliquot 350 milliliters of Opti-MEM into a sterile 500-milliliter bottle.
Add the plasma DNA for transfection into the bottle and shake to mix well. Now add 15 milliliters of PEI solution into the bottle. Shake the mixture vigorously for 30 seconds to ensure even mixing.
After incubating the mixture at room temperature for 15 minutes, transfer it to a one-liter bottle. Then add 700 milliliters of low-glucose DMEM. Remove the 10-layer Cell Factory from the incubator and vacuum the media into a waste container.
Carefully pipette the DNA transfect mixture into the Cell Factory. Then place the Cell Factory back in the incubator for 72 to 96 hours. After three to four days, filter the clarified supernatant through a 0.45-micrometer filter unit.
Add 500 milliliters of DPBS into the Cell Factory to rinse it. Centrifuge it at 4, 000 g for 20 minutes at four degrees Celsius. With a vacuum system and an aspirating pipette, remove and discard the supernatant.
Add 20 milliliters of AAV Lysis Buffer to the pellet and mix to resuspend the pellet in the buffer. Store at minus 80 degrees Celsius until purification. Now add 40%PEG sodium chloride solution to the AAV solution, making the final concentration up to 8%PEG.
Place the mixture on a low-speed orbital rotator overnight at four degrees Celsius. Centrifuge of the solution at 4, 000 g for 30 minutes at four degrees Celsius to precipitate the virus. Pipette out the supernatant.
Then had five to 10 milliliters of AAV HEPES Resuspension Buffer to the pellet. Transfer the suspension into a 50-milliliter conical tube to continue the downstream purification. Next, fix a 16-gauge punted needle fitted on a 10-milliliter syringe and overlay the prepared iodixanol gradients into a round-top polypropylene ultracentrifuge tube.
With a 22-gauge needle, carefully add up to 17 milliliters of the AAV solution on top of the gradient. Top off the tube with AAV Dialysis Buffer. Seal the tubes with an electric sealer.
Centrifuge the tubes at 350, 000 g for two hours in a titanium rotor at four degrees Celsius. Then transfer them into an ultracentrifuge tube rack. Now use a new 18-gauge needle to transfer the AAV virus into a dialysis cassette.
Remove the air from the cassette after injecting the virus into it. Place a stir bar in the beaker containing the AAV Dialysis Buffer and transfer it onto a stir plate. Then place the dialysis cassette in the buffer with a float buoy.
Use a 10-milliliter syringe to transfer the virus from the cassette into a centrifugal filter unit. Centrifuge at 4, 000 g for 15 to 30 minutes at four degrees Celsius. Collect the concentrated AAV from the filter unit.
Then rinse the filter with 300 microliters of AAV Dialysis Buffer. Finally, transfer the rinse solution to the concentrated AAV. The isolated AVV virus was found to have a purity greater than 90%rendering it suitable for in vivo usage.
